Macrophages play an essential role in host defense: they ingest and kill pathogens, process and present antigens to lymphocytes, and secrete functionally diverse cytokines. Patients lacking healthy phagocytic cells, such as macrophages and neutrophils, exhibit a lethal susceptibility to infection by microbes. Monocytes display an important cytotoxic activity for tumor cells in breast, lung, and gastrointestinal cancers. However, little is known about the molecular mechanisms controlling myeloid cell (macrophage and neutrophil) development. The proto-oncogene Spi-1 and a related gene, Spi-B, are the first examples of lineage-restricted transcription factors implicated in gene regulation in myeloid cells. Using a genetic approach, involving mouse embryonic stem cells and mutant myeloid cell lines, we will assess the role of Spi-1 and Spi-B in myeloid cell development and gene expression. We will also learn what roles they play in the production of other hematopoietic cells. Spi-1 causes erythroleukemias when aberrantly expressed in red cell precursors. We will show which known myeloid- specific genes are downstream targets for Spi-1 and ascertain if they are involved in erythroleukemogenesis. We will identify novel Spi-1-regulated genes and determine if they also function in Spi-1- induced oncogenesis. We will assess the regulation of a myeloid- specific gene, the c-fes proto-oncogene, by Spi-1 and Spi-B. We hope to identify and clone genes encoding other myeloid-specific nuclear regulatory proteins. The discovery of additional lineage- restricted myeloid transcription factors is necessary for a complete understanding of myeloid cell gene expression. These experiments are critical to a better description of the molecular events that produce myeloid cells from hematopoietic progenitor cells. The proposed studies are relevant both to normal hematopoietic (and specifically myeloid cell) development and to the molecular pathogenesis of hematopoietic neoplasia.